PWS UPD1.2 Quality Control Package rev3 - FPWR Docs/PWS UPD1.2 Validation Q… · DNA Profile...
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1 Validation Package Product Type Cell Line Name PWS UPD1.2 Cell Type Human induced pluripotent stem cell (iPSC) Donor Gender Female Source Tissue Fibroblasts Reprogramming Method Lentivirus Method: Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells. 2009;27(3):543-9. Publications Langouët M, Glatt-Deeley HR, Chung MS, Dupont-Thibert CM, Mathieux M, Banda EC, Stoddard CE, Crandall L, Lalande M; Zinc finger protein 274 regulates imprinted expression of transcripts in Prader-Willi syndrome neurons, Human Molecular Genetics, 2018;27(3):505-515 Biosafety Level 2 Thaw Recommendation Thaw 1 vial into 1 well of a 6 well plate Growth Conditions Feeder Dependent: irradiated MEF (Gibco A34181), hESC medium: DMEM/F12 (Gibco 11330-057) with 20% Knockout Serum Replacement (Invitrogen 10828-028), 1X Non-essential amino acids, 2mM L-glutamine, 0.1mM 2-Mercaptoethanol, 8ng/mL basic Fibroblast Growth Factor Passage Number 28, these cells were cultured for 28 passages prior to freeze Date Vialed September 6, 2018 Cryopreservation Bambanker (Wako Chemicals Cat. No, 302-14681) Serum-free cell freezing medium, containing 10% DMSO Storage Cryopreserved cells should be stored in liquid nitrogen Cells should be cultured at 37 °C upon arrival Shipped Frozen vials or ambient temperature as live cells in T25 flask Banked By Stem Cell Core, UConn Health
Transcript of PWS UPD1.2 Quality Control Package rev3 - FPWR Docs/PWS UPD1.2 Validation Q… · DNA Profile...
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ValidationPackage
ProductType CellLineName PWSUPD1.2CellType Humaninducedpluripotentstemcell(iPSC)DonorGender FemaleSourceTissue FibroblastsReprogrammingMethod
LentivirusMethod:SommerCA,StadtfeldM,MurphyGJ,HochedlingerK,KottonDN,MostoslavskyG.Inducedpluripotentstemcellgenerationusingasinglelentiviralstemcellcassette.StemCells.2009;27(3):543-9.
Publications LangouëtM,Glatt-DeeleyHR,ChungMS,Dupont-ThibertCM,MathieuxM,BandaEC,StoddardCE,CrandallL,LalandeM;Zincfingerprotein274regulatesimprintedexpressionoftranscriptsinPrader-Willisyndromeneurons,HumanMolecularGenetics,2018;27(3):505-515
BiosafetyLevel 2ThawRecommendation
Thaw1vialinto1wellofa6wellplate
GrowthConditions
FeederDependent:irradiatedMEF(GibcoA34181),hESCmedium:DMEM/F12(Gibco11330-057)with20%KnockoutSerumReplacement(Invitrogen10828-028),1XNon-essentialaminoacids,2mML-glutamine,0.1mM2-Mercaptoethanol,8ng/mLbasicFibroblastGrowthFactor
PassageNumber 28,thesecellswereculturedfor28passagespriortofreezeDateVialed September6,2018Cryopreservation Bambanker(WakoChemicalsCat.No,302-14681)
Serum-freecellfreezingmedium,containing10%DMSOStorage Cryopreservedcellsshouldbestoredinliquidnitrogen
Cellsshouldbeculturedat37°CuponarrivalShipped FrozenvialsorambienttemperatureaslivecellsinT25flaskBankedBy StemCellCore,UConnHealth
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Contents
TestDescription TestProvider PageCultureCharacteristics:Growth,CryopreservationandRecovery
StemCellCore,UConnHealthFarmington,CTwww.health.uconn.edu/stem-cell-core
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GeneExpression(PraderWilli)StemCellCore,UConnHealth 8-9EmbryoidBodyFormationStemCellCore,UConnHealth 10-12DNAMethylationStemCellCore,UConnHealth 13-14PluripotencyTestandLineageScore(TaqManScorecard,AppliedBiosystems)
StemCellCore,UConnHealth
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Cyto-SNPAnalysisUConnChromosomeCoreStorrs-Mansfield,CTwww.cgi.uconn.edu
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HumanPathogenTesting
IDEXXBioAnalyticsColumbia,MOwww.idexxbioanalytics.com
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DNAProfile(MatchfibroblastsampletoiPSCline)
IDEXXBioAnalytics 20
MycoplasmaTesting IDEXXBioAnalytics 20Microbiology(BacterialandFungal)Testing
IDEXXBioAnalytics 20
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CultureCharacteristics:Cryopreservation:AspirateculturemediumfromhPSCplate,washoncewithPBS.Add1mLof0.5uMEDTA(Invitrogen,15575-038)dissociationsolution,incubate3-5minutesat37°C.AspirateEDTAsolutiongently,add1mlofculturemediumperwell.CutstemcellcoloniesusingtheStemProEZPassageDisposableStemCellPassagingTool(Invitrogen,23181-010).Useacellscrapertogentlydetachthecellsoffthesurfaceofthecultureplate.Transferthemediumcontainingcoloniestoa15mltubeandspindownat1000rpm(200g)for2min.Aspiratethesupernatantcarefullytoremovesinglecellsorcontaminatingfeedercells(MEFs)fromthepopulation.Re-suspendcoloniesinBambanker(Cat.No,302-14681)serum-freecellfreezingmedium,containing10%DMSO,andplacethecellsincryogenicvialsforfreezingandpreservation.Recovery:Rollthevialbetweenglovedhandsfor3-5secondstoremovethefrost.Immersethevialintoa37°Cwaterbath.Swirlthevialgentlyandobservetheprogressofthethaw.Whenonlyasmallicecrystalremains,wipetheoutsideofthevialwith70%ethanol.Inasterilebiologicalsafetycabinet,transferthecontentsofthecryogenicvialdirectlytothebottomofa15mLconicaltube.Slowlyadd4mLofhESCmediumtothetube.Centrifugethecellsfor5minutesat200xg.GentlyresuspendthecellsinhESCmedium.AspiratethePBSfromtheMEFfeederwellandslowlyaddthecellsuspensiontothepreparedwellofthe6-wellplate.GrowthCurve:CellsfromhPSCwerepassagedusingAccutase(EMDMillipore,SCR005)for8minutes,andthenmechanicallydissociatedintosinglecellsusingpipette1000ultips.Centrifugethecellsat200xgfor5minutes.1500cellsperwellofa6-wellplatewereplatedonMEFusinghESCmedium.MCH2-10(generatedfromanunaffecteddonor)servedasacontrol.Cellsfromthreeseparatewellswereharvestedeverypassage,accutasedandcounted.
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PWS-UPD1.2beforecryopreservationPhaseimages
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PWS-UPD1.2Day5afterrecoveryfromcryopreservationPhaseimages
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GrowthCurve:coloniesinonewellofa6wellplateintriplicatewells,1500cellsplatedtoeachforbothtestandcontrol.MCH2-10wasusedasacontrol.
CultureCharacteristics-GrowthCurve:cellnumberinonewellofa6wellplate
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PWSUPD1.2 MCH2-10
ColoniesFirstPassage
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PWSUPD1.2 MCH2-10
ColoniesSecondPassage
050001000015000200002500030000
PWSUPD1.2 MCH2-10
CellNumberFirstPassage
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20000
40000
60000
80000
PWSUPD1.2 MCH2-10
CellNumberSecondPassage
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GrowthCurve:Phaseimagesfromday3afterpassage;thirdpassageofassay.
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GeneExpression-PWSchromosome15q11–q13regiongenes(qRT-PCR)
RNAwasisolatedfromiPSCcellsusingQuick-DNA/RNAMiniprepKit(ZYMOResearch,D7001).cDNAwassynthesizedusingSuperScriptIIReverseTranscriptase(Invitrogen,18064-022).GeneexpressionwasanalyzedusingTaqManGeneExpressionAssays,andtheGAPDHwasusedasanendogenouscontrol.ThedatawereanalyzedusingBio-RadCFXManager3.1software,normalizedtoMCH2-10(iPSCgeneratedfromunaffecteddonor).TheTaqmanFAM-MGBqRT-PCRprimersusedtoexaminethegeneexpressionofMKRN3,MAGEL2,NDN,SNRPN,SNORD116andIPW.
TaqManGeneExpressionassaysareusedforquantitativereal-timePCRanalysisofgeneexpressionandconsistofapairofunlabeledPCRprimersandaTaqManprobewithadyelabel(FAM)onthe5’endandaminorgroovebinder(MGB)andnon-fluorescentquencher(NFQ)onthe3’end.
Mapofchromosome15q11–q13region:
GeneSymbol TaqManAssayIDMKRN3 Hs00271653_s1MAGEL2 Hs00255922_s1NDN Hs00267349_s1SNRPN Hs00243205_m1SNORD116 Hs03454084_m1IPW Hs03455409_s1GAPDH Hs99999905_m1
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Geneexpression(PraderWilli)analysisofMKRN3,MAGEL2,NDN,SNRPN,SNORD116,IPW.GADPHwasusedasanendogenouscontrolanddatawerenormalizedtoMCH2-10.
AS2.1isaniPSClinegeneratedfromadonorwithAngelmanSyndrome*PWS1.7isaniPSClinegeneratedfromadonorwithPraderWilliSyndrome(LargeDeletion)*MCH2-10isaniPSCgeneratedfromanunaffecteddonor**ChamberlainSJ,ChenPF,NgKY,etal.InducedpluripotentstemcellmodelsofthegenomicimprintingdisordersAngelmanandPrader-Willisyndromes.ProcNatlAcadSciUSA.2010;107(41):17668-73.
0.000
0.500
1.000
1.500
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
MKRN3
0.000
0.400
0.800
1.200
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
MAGEL2
0.000
0.400
0.800
1.200
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
NDN
0.000
0.400
0.800
1.200
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
SNRPN
0.000
0.400
0.800
1.200
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
SNORD116
0.000
0.400
0.800
1.200
PWSUPD1.2
PWS1.7 AS2.1 MCH2-10
IPW
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Embryoidbodies(EB)arethethree-dimensionalaggregatesformedinsuspensionbytheiPSCs.EmbryoidBodycultureisusedtoexaminethedifferentiationpotentialoftheiPSCs.Growthanddifferentiationofembryoidbodies:aspirateofftheculturemediumfromthecultureplates,andthenadd1mLpre-warmedEBmedium(hESCmediumlackingbasicfibroblastgrowthfactor)toeachwellof6-wellplate.CutstemcellcoloniesusingtheStemProEZPassagedisposablestemcellpassagingtool(Invitrogen,23181-010).Useacellscrapertogentlydetachthecellsoffthesurfaceofthecultureplate.Gentlytransferthecellclumpsintoa15-mLconicaltube.Allowthecellstogravitysedimentforapproximately5minutes.Aspiratethesupernatant,andthengentlytapthetubetoloosenthecellpellet.Transferthecellclumpstoacorningultra-Lowattachmentcellcultureflask(Sigma,CLS3815)inatotalof10mLofEBmedium.Replacedmediumandtookimageeveryotherday.RNAwascollectedatday14fortri-Lineagedifferentiationassay.
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PWSUPD1.2embryoidbodyformation(1of2)PhaseImagesDay0today14
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PWSUPD1.2embryoidbodyformation(2of2)PhaseImagesDay0today14
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DNAMethylationanalysisofPWS-ICusingamethylation-sensitiverestrictionendonucleasequantitativePCRassay.TheEpiTectIIDNAMethylationEnzymeKit(Qiagen,335452)preparesgenomicDNAsamplesforDNAmethylationanalysisusingEpiTectMethylIIPCRAssaysforindividualandpredictedmethylatedCpGislands.Usingtheenzymesandbufferprovidedinthekit,4digestsareperformedtodetectdifferentmethylatedDNAfractions.Theproductofamockdigest(Mo)containsalloftheinputgenomicDNA.Theproductofthemethylation-sensitiverestrictionenzymemixture(EnzymeA)digest(Ms)containsmethylatedDNAsequences,whiletheproductofthemethylation-dependentrestrictionenzymemixture(EnzymeB)digest(Md)containsunmethylatedDNAsequences.Theproductofadoubledigest(Msd)measuresthebackgroundandthesuccessofbothenzymaticdigestions.
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DNAMethylationanalysisofPWS-ICusingamethylation-sensitiverestrictionendonucleasequantitativePCRassay.CellLine Unmethylated MethylatedPWSUPD1.2 1.71% 98.29%PWS1.7 2.39% 97.61%AS2.1 92.61% 7.39%MCH2-10 40.96% 59.04%H9 52.11% 47.89%
AS2.1isaniPSClinegeneratedfromadonorwithAngelmanSyndrome*PWS1.7isaniPSClinegeneratedfromadonorwithPraderWilliSyndrome(LargeDeletion)*MCH2-10isaniPSCgeneratedfromanunaffecteddonor*H9hESCisfromWiCellResearchInstitute,Madison,WI*ChamberlainSJ,ChenPF,NgKY,etal.InducedpluripotentstemcellmodelsofthegenomicimprintingdisordersAngelmanandPrader-Willisyndromes.ProcNatlAcadSciUSA.2010;107(41):17668-73.
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ScorecardPluripotencyandTri-LineageDifferentiationAssayTaqManhPSCScorecardPanel384-well(AppliedBiosystems,A15870)enablesverificationofpluripotencyanddeterminationoflineagebiasforiPSCcellline.The384-wellplatecontainsfoursetsof94predefinedTaqManGeneExpressionassays(includingendogenouscontrols)dried-downinthewells.TheScorecardrunonthe7900HTReal-TimePCRSystem.ThedatawereanalyzedusingAppliedBiosystemshPSCScorecardanalysissoftware.Scorecard:Asimple-to-interpretsummaryofgeneexpressionleveldatathatconfirmspluripotencyorindicategermlayerbiasofyoursample.HeatMap:Colorsindicatethefoldchangeinexpressionrelativetotheundifferentiatedreferencesetforeachgene.ScoresBox&WhiskerPlot:Viewsamplesscores(color)inrelationtotherangeofscoresfortheundifferentiatedreferenceset(gray).CorrelationPlot:Seehowgeneexpressionlevelscorrelatebetweensamples.AssayQC:Performaquickqualitycontrolchecktomakesurethesampleamplifiedasexpected.
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Cyto-SNP
TheAffymetrixCytoScanHDArrayincludes750,000SNPsand2.6millioncopynumbermarkerstoenabledetectionofaccuratebreakpointassignmentandhigh-resolution(~25kbresolution)detectionofcopynumbervariation(CNV),lossofheterozygosity(LOH),uniparentaldisomy(importantforimprintingsyndromestudies)andlow-levelmosaicismincelllines.
• Toidentifychromosomeabnormalitiesatlessthan5MBresolution• ToconfirmG-bandandFISHfindings• Todefinespecificbreakpointsand/orgeneinsertions• WhenLOHand/orCNVanalysesareneeded• Toidentifyamplificationsordeletionsforgenesofinterest• Whenwholegenomegenotypingisneeded• Toderivegenomicinformationonsubtelomericandpericentromericregions
GenomicmicroarrayanalysisandG-bandedkaryotypingarecomplementaryandprovideacomprehensivepanelofgenomeintegrityassessment.
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Sample ID: CC18-30Sample Name: PWS_UPD 1.2
Experiment date:Report date:
Microarray type:Microarray Barcode:
SNP manifest file:Annotation DB:SNP cluster file:
Genome build name: GRCh37GTC file:
Algorithm:Smoothing:
CGH Reporting:
Significant Clones:QC Measures:
Median Log R Deviation: Ratio Intensity Signal (<0.2)Median Call Rate: genotype calling performance estimate (>0.98 )
Sample sex:
ISCN Type Chromosome End15q21.1-15q21.1 LOH 15 48,957,597
15q26.2-15q26.3 LOH 15 102,376,655
17q21.31-17q25.3 LOH 17 81,060,040
Array processing: Lisa LaBelle, MS, MB (ASCP)Data analysis and sign out: Judy Brown, PhD, CG, MB (ASCP)
Backbone = 9Minimum Del and Dup Size = 600 KbMinimum LOH Region Size (Mb) = 3.0CGH Region = 10 LOH Region = 500
BG_Annotation_Ens74_20180801.dbCytoSNP-850Kv1-Ensembl version: 74202917700009_R01C01.gtcBeadArray v2 - Standard
Case Report
December 6, 2018 Illumina CytoSNP-850K v1.2202917700009CytoSNP-850Kv1-2_NS550_B3.bpm
November 5, 2018
Warning: The results reported herein are for research use only and not to be used for patient diagnosis or treatment.
The significance of the Illumina molecular karyotyping findings should be interpreted by the principle investigator for research purposes only and include consideration of cell origin, culture conditions and experimental questions. LogR changes and B-allele frequencies were manually scanned across all chromosomes. Gains and losses at 400 Kb or larger and LOH at 5 Mb are reported . Chromosome 15 was evaluated separately. Please note: loss of heterozygosity was NOT identified for the entire length of the long arm of chromosome 15. The PWS critical region (15q11.2-q13) is reported as heterozygous. No deletions or duplications were detected.
Pass0.181
Female
Loss of heterozygosity on long arm chromosome 15 of 0.58 Mb (<1 Mb) ; overlaps 6 HGNC and 4 OMIM gene(s).
Loss of heterozygosity on long arm chromosome 17 of 39.614896 Mb ; overlaps 698 HGNC and 383 OMIM gene(s).
Loss of heterozygosity on long arm chromosome 15 of 6.99 Mb ; overlaps 54 HGNC and 16 OMIM gene(s).
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